Rickettsia rickettsii and Coxiella burnetii are the etiologic agents of Rocky Mountain spotted fever and human Q fever, respectively. Due to an obligate intracellular nature and the lack of workable genetic tools, our current knowledge of specific mechanisms of Rickettsia and Coxiella pathogenesis is limited. Moreover, critical specific and non-specific host defenses that are necessary for controlling infection are ill defined. Nitric oxide (NO) is a critical signaling molecule and responsible for inhibiting the growth of a number of intracellular pathogens. Inducible nitric oxide synthase (iNOS)-dependent high-output production of NO is observed in a number of cells types and can be stimulated by proinflammatory cytokines and bacterial products such as lipopolysaccharide. The roles of nitric oxide and reactive nitrogen intermediates (RNI) in controlling infection by C. burnetii and R. rickettsii are unknown. The long-term objective of this research is to understand the role of NO in the infectious cycle of C. burnetii and R. rickettsii. in addition to utilizing conventional NO probes, this research will employ the novel probes being developed in Project 1 of this NIH IDeA COBRE proposal that afford fine spatiotemporal measurement and release of NO. To achieve our objective, Specific Aim 1 will qualitatively assess the level of NO production in murine L-929 fibroblast cells infected with virulent or avirulent strains of C. burnetii and R. rickettsii, and determine if production is cytokine and iNOS dependent. Specific Aim 2 will determine whether NO inhibits and/or clears infection by virulent and avirulent strains of R. rickettsii and C burnetii. The effect of local NO production on infection will be conducted by seeding cell monolayers over NO-releasing micro spheres. The effect of system-wide release of NO on killing R. rickettsii and C burnetii will be investigated by subjecting infected cells to NO stress by exposure to DETA NONOate, or iNOS-inducing cytokines. Specific Aim 3 will quantify the dose of NO required for intracellular killing of virulent and avirulent strains of R. rickettsii and C. burnetii. Concentration versus time profiles of NO release from the extracellular NO donor DETA NONOate will be established with a NO- detecting electrochemical probe and a NO dose-killing response curve will be established for each organism. Lastly, Specific Aim 4 will determine the effects of NO on morphological differentiation and actin-based motility (ABM), virulence mechanisms employed by C. burnedi and R. rickettsii, respectively. Moreover, pathogen proteins that are up-regulated in response to NO/RNI stress will be identified and their encoding genes cloned. The encoded proteins will be over-expressed and purified for biochemical studies, and genes tested in a heterologous microbial system for their ability to confer resistance to NO/RNI. Not only will these studies lead to a greater understanding of the role of NO in Rickettsia and Coxiella pathogenesis, they will also closely integrate with the studies proposed by co-investigators within this NIH IDeA COBRE.